Method of peptide synthesis

ABSTRACT

The present invention relates to a process for forming an N-α-amino protected amino acid fluoride in situ by reacting an N-α-amino protected amino acid with an ionic fluoride salt in the presence of a peptide coupling agent. It is also directed to the use of the amino acid fluoride thus formed in peptide synthesis.

This application is a divisional of Ser. No. 08/588,187 (Jan. 18, 1996)now U.S. Pat. No. 5,849,954.

GOVERNMENT SUPPORT

This work has been supported by grants from the National Institutes ofHealth GM-09706 and the National Science Foundation (CHE 9314083). TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a new process for effecting peptidesynthesis. More specifically, it relates to a new process for preparingan amino acid fluoride, and also to the use thereof in peptidesynthesis. Furthermore, the present invention relates to new reagentsfor preparing the amino acid fluorides.

2. Background of the Invention

Amino acid fluorides have been shown to be a convenient and highlyefficient reagent for both solution and solid phase peptide synthesis.When used in peptide synthesis, the peptide formed therefrom is producedin high yield and relatively pure form, with minimal racemization.Furthermore, it has been shown that there are many other advantagesassociated with the use of amino acid fluorides in peptide synthesis.For example, the acid fluorides allow the syntheses of peptides whichincorporate highly-hindered amino acids, such as α-amino isobutyric acid(Aib) and α-ethylalanine (e.g., isovaline, Iva), and the like.Furthermore, amino acid fluorides exhibit several advantages relative toother amino acid halides, e.g., amino acid chlorides or bromides, in thecoupling reaction and formation of peptides. For example, unlike theother amino acid halides, amino acid fluorides can accommodate t-butylside chain protecting groups. Moreover, conversion to the correspondingoxazolone in the presence of t-organic base does not occur, thusavoiding the danger of racemization. Furthermore, the coupling reactionsoccur readily in the complete absence of an organic base, again avoidingpossible racemization.

Moreover, another advantage of amino acid fluorides is that they areeasily synthesized from the corresponding amino acid and are isolable incrystalline form. They are generally stable and have a long shelf life.

In view of these advantages, it is highly desirable to utilize aminoacid fluorides in peptide synthesis. Thus, peptide synthesis can beeffected by first preparing a N-α-amino protected amino acid fluorideand then utilizing this amino acid fluoride as a coupling agent toproduce the desired peptide.

Unfortunately, when employing FMOC amino acid fluorides in practicalpeptide synthesis, difficulties were encountered in the case of twoamino acids, arginine and histidine. In the latter case, while FMOC—His(Trt)—F has been synthesized and used in coupling reactions, its longterm shelf stability is in doubt. For sulfonamide-protected argininederivatives (e.g., FMOC—Arg (Pbf)—OH or FMOC—Arg—(Pmc)—OH)), thecorresponding acid fluorides could not be synthesized due to theirfacile cyclization to the corresponding lactam.

Thus, investigations were conducted to improve the efficiency thereofand to overcome these problems. It was felt that the efficiency of theoverall process would be enhanced if the amino acid fluoride wereproduced in situ. Thus, Carpino et al., as described in JACS 95, 117,5401, developed a new uronium-style reagent TFFH, 1, which

has been shown to act as a coupling reagent which acts via in situconversion to an acid fluoride:

Although a useful technique, especially since TFFH is relativelyinexpensive, a disadvantage of this method is the need to use a basicreagent, such as N,N-diisopropylethylamine (DIEA) in the activation step(Eq. 1). Indeed, the speed of conversion of the acid to the acidfluoride increases with the number of equivalents of DIEA used (1 eq<<2eqs<3 eqs<4 eqs).

Although this technique was more efficient than methods heretofore usedin peptide coupling, scientific investigations were conducted to improveupon this reagent. It was believed that the in situ process for thepreparation of protected amino acid fluorides and the overall process ofpeptide synthesis would be improved if a method could be found togenerate amino acid fluorides in situ without the presence of a basicreagent. The present inventors have found such a method. Moreover, thepresent in situ process overcomes the difficulties discussed hereinabovewith histidine and arginine.

SUMMARY OF THE INVENTION

Accordingly, the present invention is directed to a process of preparinga N-α-amino protected amino acid fluoride in situ by reacting aN-α-amino protected amino acid or acylating derivative thereof in thepresence of a coupling agent with an ionic fluoride salt. In a preferredembodiment, the anion has the formula:

Ly(HF)_(z)F⁻ or TG₁G₂G₃F₂ ⁻  I

wherein

z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

y is 0 or 1;

L is TG₁G₂G₃G₄;

T is a Group IV element consisting of Si,

Ge, Sn and Pb;

G₁, G₂, G₃ and G₄ are independently halogen, hydrogen, alkyl, aryl, arylalkyl, cycloalkyl or cycloalkyl alkyl.

The present invention is also directed to the preparation of peptidesfrom the in situ preparation of acid fluorides in accordance with theprocedure described hereinabove. Finally, the present invention isdirected to the novel fluoride salts useful in the in situ preparationof amino acid fluorides.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As indicated hereinabove, an aspect of the present invention is directedto the in situ synthesis of an N-α-amino protected amino acid fluoridefrom an N-α-amino protected amino acid or acylating derivative thereofand an ionic fluoride salt in the presence of a coupling agent.

The term “amino acid” is a term of art that is readily understood by theskilled artisan.

As used herein, the term “amino acid” refers to an organic acidcontaining both a basic amino group (NH₂) and an acidic carboxyl group(COOH). Therefore, said molecule is amphoteric and exists in aqueoussolution as dipole ions. (See, “The Condensed Chemical Dictionary”, 10thed. edited by Gessner G. Hawley, Van Nostrand Reinhold Company, London,Eng. p.48 (1981)). The preferred amino acids are the α-amino acids. Theyinclude, but are not limited to the 25 amino acids that have beenestablished as protein constituents. They must contain at least onecarboxyl group and one primary or secondary amino group on the aminoacid molecule. They include the naturally occurring amino acids. Forexample, they include such proteinogenic amino acids as alanine, valine,leucine, isoleucine, norleucine, proline, hydroxyproline, phenylalanine,tryptophan, methionine, glycine, serine, threonine, cysteine, cystine,tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,hydroxylysine, ornithine, arginine, histidine, naphthylalanine,penicillamine, β-alanine, isovaline, α-amino isobutyric acid, and thelike.

A N-α-amino protected amino acid designates an amino acid in which theα-amino group contains a blocking group. These type of blocking groups(also designated as protecting groups) are well known in the art.Examples include 9-fluorenylmethyloxy-carbonyl(FMOC), 2-chloro-1-indanylmethoxy carbonyl (CLIMOC), and benz-[f]-indene-3-methyloxycarbonyl(BIMOC) and dbd-TMOC which are discussed in U.S. Pat. Nos. 3,835,175,4,508,657, 3,839,396, 4,581,167, 4,394,519, 4,460,501 and 4,108,846, andthe contents thereof are incorporated herein by reference as if fullyset forth herein. Moreover, other amino protecting groups such as2-(t-butyl sulfonyl)-2-propenyloxycarbonyl (Bspoc) and benzothiophenesulfone-2-methoxycarbonyl (Bsmoc) are discussed in copendingapplication, U.S. patent application Ser. No. 364,662 and the subjectmatter therein is incorporated herein by reference. Other aminoprotecting groups include those described in an article entitled “SolidPhase Peptide Synthesis” by G. Barany and R. B. Merifield in Peptides,Vol. 2, edited by E. Gross and J. Meienhoffer, Academic Press, New York,N.Y., pp. 100-118 (1980), the contents of which are incorporated hereinby reference. These N-amino protecting groups include such groups asFMOC, Bspoc, Bsmoc, t-butyloxycarbonyl (BOC), t-amyloxycarbonyl (Aoc),β-trimethylethyloxycarbonyl (TEOC), adamantyloxycarbonyl (Adoc),1-methylcyclobutyloxycarbonyl (Mcb),2-(p-biphenylyl)propyl-2-oxycarbonyl (Bpoc),2-(p-phenylazophenyl)propyl-2-oxycarbonyl (Azoc),2,2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl (Ddz),2-phenylpropyl-2-oxycarbonyl (Poc), benzyloxycarbonyl (Cbz),p-toluenesulfonyl aminocarbonyl (Tac), o-nitrophenylsulfenyl (Nps),dithiasuccinoyl (Dts), phthaloyl, piperidino oxycarbonyl, formyl,trifluoroacetyl and the like.

The preferred protecting groups can be placed into five categories:

1) a base labile N-α-amino acid protecting group such as FMOC, and thelike.

2) protecting groups removed by acid, such as Boc, TEOC, Aoc, Adoc, Mcb,Bpoc, Azoc, Ddz, Poc, Cbz, 2-furanmethyloxycarbonyl (Foc),p-methoxybenzyloxycarbonyl (Moz), Nps, and the like.

3) protecting groups removed by hydrogenation such as Dts, Cbz.

4) protecting groups removed by nucleophiles, such as Bspoc, Bsmoc andNps and the like.

5) protecting groups derived from carboxylic acids, such as formyl,acetyl, trifluoroacetyl and the like, which are removed by acid, base ornucleophiles.

It will be apparent to one skilled in the art, that in the course ofprotein synthesis, it may be necessary to protect certain side chains ofthe amino acids to prevent unwanted side reactions. For example, it maybe necessary to protect the hydroxyl group on the side chain oftyrosine, serine, or threonine in order to prevent these groups frominterfering with the desired reactions. This is a common problem inpeptide synthesis and many procedures are available for protecting thefunctional groups on the side chains of the amino acids. Such proceduresfor protecting various functional groups are known to one skilled in theart and are described in the treatise entitled “The PEPTIDES”, Vol. 2,Edited by E. Gross and J. Meienhoffer, Academic Press, NY, N.Y., pp.166-251 (1980), and the book entitled “Protective Groups in OrganicSynthesis”, by T. W. Green, John Wiley and Sons, New York, 1981, thecontents of both being incorporated herein by reference.

The various protecting groups for the N-α-amino group and side chainsare described in U.S. Pat. No. 5,360,928 and copending applicationentitled “Cyclopropyl Based O and N and S-Protecting Groups” havingSerial Number U.S. Ser. No. 08/221,226, assigned to the same assignee asthat of the present application. The contents of both are incorporatedherein by reference as if fully set forth herein.

Thus, the term “N-α-amino protected amino acid” encompasses those aminoacids having a protecting group on the α-amino group. The side chainthereof may or may not have a blocking group; it may not be necessary,as with some amino acids which do not have on the side chain afunctional group, i.e., a group which is reactive with the reagents orproducts formed under peptide forming conditions if not protected by ablocking group. The amino acids which do not have a functional groupthereon include such amino acids as glycine, alanine, valine and thelike. On the other hand, the side chain may have a functional group, butit may or may not be protected. However, if being used in peptidesynthesis, it is preferred that the functional group be protected by aside chain blocking group.

An acylating derivative of said amino acid refers to an acylating groupthat replaces the OH group on the α-carboxyl group. Examples includeacid halides, i.e., Br, I or Cl; esters, such as aryl, alkyl, arylalkyl,cycloalkyl or cycloalkyl alkyl esters; alkyl anhydrides and the like.

A peptide coupling agent is another term of art readily understood bythe skilled artisan. They include the dehydrating agents normally usedin peptide formation. Examples of coupling agents are carbodiimides,such as N,N-dicyclohexylcarbodiimide (DCC), N,N-Diisopropylcarbodiimide(DIC), N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC), and thelike, the active esters, such as esters of 1-hydroxybenzotriazole(HOBt), N-ethyloxycarbony-2-ethyloxy-1,2-dihydroquinone (EEDQ), propanephosphonic acid anhydride (T3P) etc. Other coupling agents are describedin copending application entitled “New Reagents For Peptide Couplings”,having U.S. Ser. No. 08/127,675, which has been assigned to the presentassignee, the contents of which are incorporated by reference.

The aforementioned application discloses coupling agents of the formula:

and N-oxides thereof and salts thereof wherein R₁ and R₂ taken togetherwith the carbon atoms to which they are attached form a heteroaryl ringwherein said heteroaryl ring is an oxygen, sulfur or nitrogen containingheteroaromatic containing from 3 and up to a total of 13 ring carbonatoms, said heteroaryl may be unsubstituted or substituted with loweralkyl or an electron-donating group;

Y is O, NR₄, CR₄R₅;

R₄ and R₅ are independently hydrogen or lower alkyl;

X is CR₆R₇ or NR₆;

R₆ and R₇ are independently hydrogen or lower alkyl; or R₆ and R₇ takentogether form an oxo group or when n=O, R₄ and R₆ taken together mayform a bond between the nitrogen or carbon atom of Y and the nitrogen orcarbon atom of X;

Q is (CR₈R₉) or (NR₈);

when n is 1, R₄ and R₈ taken together may form a bond between the ringcarbon or nitrogen atom of Q and the ring carbon or nitrogen atom of R₈;

each n is independently 0, 1 or 2;

R₃ is lower alkyl carbonyl, aryl carbonyl, lower aryl alkyl carbonyl, apositively charged electron withdrawing group, SO₂R₁₄ or

R₁₄ is lower alkyl, aryl or lower arylalkyl;

q is 0-3;

R₈ and R₉ are independently hydrogen or lower alkyl or R₇ and R₈ takentogether with the carbon to which they are attached form an aryl ring,and m is 0 or 1.

Preferred embodiments for R₃ include lower alkyl carbonyl, arylcarbonyl, lower aryl alkyl carbonyl and a positively charged electronwithdrawing group. It is preferred that R₃ is not a phosphonium cation.

The term “electron withdrawing groups” as defined herein refers to agroup that will draw electrons to itself more than a hydrogen atom wouldif it occupied the same position in the molecule. S, J. March, AdvancedOrganic Chemistry, 3rd Ed., John Wiley & Sons P. 17 (1985). They includesuch groups as nitro, monohaloalkyl, dihaloalkyl, trihaloalkyl (e.g.,CF₃), halo, formyl, lower alkanoyl, lower alkylsulfonyl, loweralkylsulfonyl, and the like.

A positively charged electron withdrawing group is an electronwithdrawing group bearing a positive charge and forming a stable bond toa N-hydroxide (N—O). These types of groups are well known in the art.Examples include uronium groups, e.g.,

imino cations e.g.,

and the like, wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently hydrogenor lower alkyl, lower alkoxy lower alkyl or R₁₀ and R₁₂ taken togetherwith the atoms to which they are attached form a ring containing up to 6ring atoms and up to a total of 5 ring carbon atoms or R₁₂ and R₁₃ orR₁₀ and R₁₁ taken together with the nitrogen atom to which they areattached may form a 5 or 6 membered heterocyclic ring containing up to atotal of 5 ring carbon atoms. The heterocyclic ring thus formedpreferably contains one nitrogen atom. It is preferred that R₁₀ and R₁₁and R₁₂ and R₁₃, when both are present, are the same. It is especiallypreferred that R₁₀, R₁₁, R₁₂, R₁₃, whenever are present, are the same.

Preferred cyclic uronium and imino groups have the formula

respectively, wherein R₁₁ and R₁₂ are as defined hereinabove and M₂ is 0or 1.

It is preferred that compounds of Formula II have the formula:

wherein R₁, R₂, R₃, Y and X are as defined hereinabove.

Other preferred compounds of Formula II have the formula:

or N-oxides thereof

wherein Q, Y, X, R₃, n, R₄, R₅, R₆, R₇, R₈, R₉ and R₁₄ are as definedhereinabove,

A is N or CR₁₅;

D is CR₁₆ or N;

E is CR₁₇ or N;

G is CR₁₉ or N; and

R₁₅, R₁₆, R₁₇ and R₁₈ are independently hydrogen or lower alkyl or anelectron donating group or R₁₆ and R₁₇ taken together form an aryl ring,but at least one of A, D, E, G is N.

It is preferred that no more than two of A, D, E, G are N. It is mostpreferred that only one of A, D, E, G is N. Further, it is preferredthat R₁₅, R₁₆, R₁₇ or R₁₈ are hydrogen or an electron-donating group, asdefined herein. The preferred electron donating groups are lowerdialkylamino, especially N, N-dimethylamino and lower alkoxy, e.g.methoxy.

Preferred compounds of Formula IIA have the formulae:

or N-oxides thereof

wherein Y, X, n, Q and R₃ are as defined hereinabove and R₁₅ and R₁₇ areindependently lower alkyl and more preferably hydrogen or an electrondonating group.

Preferred compounds of Formula II also have the formula

or N-oxides thereof

wherein R₈, R₉, n, Q, D, E, X and Y are as defined hereinabove and J isNR₁₅, O, CR₁₅R₁₉ or S(O)p, and p is 0, 1, 2.

R₁₅ is as defined hereinabove and R₁₉ is hydrogen or lower alkyl. It ispreferred that R₁₉ is hydrogen, and preferred values of R₁₅ are anelectron donating group or hydrogen.

Preferred values of J are O or S(O)p; the preferred value of p is 1.

Preferred compounds of Formula VII have the formula:

or N-oxides thereof

wherein J, Y, R₈, R₉ n and R₃ are as defined hereinabove and X is C═O.

In compounds VII, VIII or VIIa as depicted above, it is preferred thatat least one of D, E or J is a heteroatom. Furthermore, it is mostpreferred that at most two of J, E and D are heteroatoms. It is mostpreferred that only one of J, E and D is a heteroatom.

Preferred compounds have the formula:

or N-oxides thereof

wherein A, D, E, G, Y, X, R₃ and J are as defined hereinabove; and

or N-oxides thereof.

Preferred embodiments of compounds of Formula II include

or the N-oxides thereof

wherein

R₃ is

R₁₀ and R₁₂ are independently methyl, ethyl, propyl, butyl, pentylCH₂CH₂O—CH₂CH₃,

R₁₅ is Me, Et, is—Pr, iPr₂N, or CMe₃

J is O, or S(O)p, and

p is 0, 1 or 2.

Of course, various combinations and permutations of the formulaedescribed herein are also contemplated by the present invention. Inaddition, Markush groupings containing less than all of the elementsdescribed hereinabove as well as the various permutations thereof arealso contemplated by the present invention.

These coupling agents of Formula II either are known compounds areprepared in accordance with the procedure described in U.S. Ser. No.08/127,675, the contents of which are incorporated by reference.

Other coupling agents include the aryl fused compounds of the formula II

wherein Y, Q, N, and R₃ are as defined hereinabove, and R₁ and R₂ takentogether with the carbon atoms to which they are attached form arylgroups. These compounds are prepared in a manner similar to thatdescribed for the compounds in U.S. Ser. No. 08/127,675.

Additionally, the term “coupling agent” includes the mixed anhydrides ofN-α-amino protected amino acids. By definition an acid anhydride is achemical compound derived from an acid by elimination of water. A mixedanhydride is a chemical compound derived from two different acids. Asused herein, a mixed anhydride of an N-α-amino protected amino acid isthe reaction product derived from the reaction of a N-α-amino protectedamino acid and a hydrocarbyl- or hydrocarbyloxy carboxylic acid undersufficient conditions to eliminate water and to form an anhydride. It isto be noted that the anhydride is formed from the reaction of theα-carboxy group of the amino acid and the carboxy group of thecarboxylic acid. A hydrocarbyl radical, as used herein, is a radicalcontaining only hydrogen and carbon. It preferably contains 1-20 carbonatoms. Examples include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, andthe like. It is preferred that alkyl is lower alkyl, alkenyl and alkynyleach contain 2-6 carbon atoms, aryl is phenyl and cycloalkyl contains 5or 6 ring carbon atoms. It is most preferred that the mixed anhydride isderived from the reaction of the N-α-amino protected amino acid and acarbonic acid or hydrocarbyl mono-ester of carbonic acid, especiallylower-alkyl ester thereof, such as ethyl carbonic acid, isopropylcarbonic acid, sec-butyl carbonic acid, and the like. The most preferredhydrocarbyl esters of carbonic acid are alkyl esters, especially loweralkyl esters. It is also preferred that the mixed anhydride is derivedfrom the reaction of the N-α-amino protected amino acid and the monoamide of carbonic acid, including the N-alkyl and the N,N-dialkylderivatives thereof. In addition, it is preferred that the hydrocarbylcarboxylic group, is disubstituted on the α-carbon to the carboxy group.It is most preferred that the α-carbon is a tertiary carbon, e.g.,pivalic acid (trimethylacetic acid), and the like.

Besides being a coupling agent, the mixed anhydride of N-α-aminoprotected amino acids especially the hydrocarbyl esters of carbonic acidalso reacts directly with the ionic fluoride salt as defined hereinaboveand can thus forms the N-α-amino protected amino acid fluoride in situ,which can then react with a carboxy protected amino acid or peptide asdescribed herein.

In addition, the term coupling agents include the active esters,especially active esters of N-∝-protected amino acids. Examples includethe N-hydroxy-piperidine esters of the N-α-protected amino acids,N-hydroxy succinimide esters of N-α-protected amino acids and theN-α-hydroxy phthalimide esters of N-α-amino protected amino acids andthe like. It is to be noted that the esters are formed from the reactionof the ∝-carboxy groups of the amino acids and the OH groups on thephthalimide, succinimide or piperidine compounds described above underesterifying conditions. These active agents and others are described inPROTECTIVE GROUPS in Organic Synthesis by J. W. Green, John Wiley &Sons, New York, 1981, pp. 180-184, the contents of which areincorporated by reference. In addition, also preferred are aryl estersof N-∝-amino protected amino acids, wherein the aryl group isunsubstituted and more preferably substituted with electron withdrawinggroups. It is preferred that the aryl group is substituted by 1 to 5electron withdrawing groups. The preferred aryl group is phenyl andpreferred electron withdrawing groups are halo especially fluoro, nitro,alkanoyl, formyl, and the like. Examples of these active esters are thepentafluorophenyl ester of N-α-amino protected amino acid, nitrophenylester of N-α-amino protected amino acid and the like.

These esters are generated by reacting the arylol group, e.g., phenol,which is unsubstituted or substituted with electron withdrawing groupswith the ∝-carboxy group of the N-α-amino protected amino acid underesterifying conditions.

Another active ester is the ester of formula II, wherein Y, Q, X and nare as defined hereinabove, R₁ and R₂ taken together with the carbonatoms to which they are attached form an aryl or heteraryl ring whereinsaid heteraryl ring is an oxygen, sulfur or nitrogen containingheteroatom containing from 3 and up to a total of 13 ring carbon atomssaid heteroaryl may be unsubstituted or substituted with lower alkyl oran electron donating group, and R₃ is BLK₁-AA₁.

BLK₁ is an amino protecting group and AA₁ is an amino acid less ahydrogen on the N-terminus and an OH on the C-terminus, i.e., BLK₁-AA isan N-∝-amino protected amino acid as defined herein. The preferredstructures are again as defined by structures IIA-VIII as definedhereinabove and the other preferred embodiments as describedhereinabove, except that R₃ is BLK₁-AA₁.

However, as with the mixed anhydride, the active esters also reactdirectly with the ionic fluoride salt as defined herein and thus formthe N-α-amino protected amino acid fluoride in situ which can then reactwith a carboxy protected amino acid or peptide, as described herein.

Preferred coupling agents are DCC, DIC,O-benzotriazolyl-1-yl-1,1,3,3-tetramethyluroniumhexafluorophosphate(HBTU),O-(7-azabenzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate(HATU); O-(7-azabenzotriazol-1-yl-1,1,3,3-bis (tetramethylene uroniumhexafluorphosphate (HApyU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(pentamethylene) uronium hexafluorophosphate(HApipU),O-(7-azabenzotrizol-1-yl)-1,3-dimethyl-1,3-trimethylene uroniumhexafluorophosphate (HAMTU), benzotriazolyl-yl-1,1,3,3-bis(tetramethylene uronium tetrafluoroborate)(TBTU), TFFH, mixedanhydrides, EZDQ, active esters, such as pentafluorophenyl orsuccinimide esters of N-α-amino protected amino acid, and the like.

The ionic fluoride salts are salts containing a cation and an anion. Theanion portion of the salt is the portion of the salt that is involved inthe reaction for forming the amino acid fluoride. As such, it containsat least one ionizable fluoride, i.e., a fluoride ion that dissociatesfrom the salt when placed in aqueous solution. Preferred anions have theformula given herein above:

Ly(HF)_(z) F^(⊖) or TG₁G₂G₃F₂ ⁻

wherein

z is 0-10;

y is 0 or 1;

L is TG₁G₂G₃G₄;

T is a Group IV element consisting of Si, Ge, Sn or Pb; and

G₁, G₂, G₃ and G₄ are independently halogen, hydrogen, alkyl, aryl,arylalkyl, cycloalkyl or cycloalkyl alkyl.

The preferred value of Z is 0-4. It is preferred that G₁, G₂, G₃ and G₄are alkyl, aryl, or arylalkyl. It is preferred that T is Sn or Si.

Although the cation portion is a spectator ion and is not involved inthe overall reaction, certain cations are preferred. They include thealkali metals, the alkaline earth metals, hydrogen cation, ammonium(NH₄+), NQ₅Q₆Q₇Q₈+, PQ₅Q₆Q₇Q₈+, SQ₅Q₆Q₇+, HalQ₅Q₆₊, or

wherein

Q₅, Q₆, Q₇ and Q₈ are independently hydrogen, lower alkyl, aryl, or aryllower alkyl,

Hal is halo, such as iodo, bromo, or chloro.

It is preferred that Q5 is arylalkyl, especially benzyl and Q6, Q₇ andQ₈ are aryl, especially phenyl.

Preferred examples of ionic fluoride salts include (C₆H₅)₄ P^(⊕)H₂F₃^(⊖), n Bu₄N^(⊕) (C₆H₅)₃ SnF₂ ^(⊖), benzyltriphenylphosphoniumdihydrogen trifluoride, i.e., [(BTPP)H₂F₃], n-Bu₄N^(⊕)(C₆H₅)₃ SnF₂ ^(⊖),Et₄N^(⊕)F^(⊖), Me₄N^(⊕)F^(⊖), Et₂NSF₃, (Me₂N)₃S^(⊕) Me₃SiF₂ ^(⊖),Bu₄N^(⊕)F^(⊖), Bu₄N^(⊕)HF₂ ^(⊖), Me₃S^(⊕)F^(⊖), Bu₄P^(⊕)F^(⊖),Bu₄P^(⊕)HF₂ ^(⊖), Bu₄P^(⊕)H₂F₃ ^(⊖), hexadecyl N^(⊕)Me₃F^(⊖), hexadecylN^(⊕)Me₃ HF₂ ^(⊖), hexadecyl NMe₃ ^(⊕)HF₃ ^(⊖), (C₆H₅)₄P^(⊕)F^(⊖),(C₆H₅)₄P^(⊕)HF₂, (C₆H₅)₄P^(⊕)H₂F₃ ^(⊖), (C₈H₁₇)₄N^(⊕)F^(⊖),(C₈H₁₇)₄N^(⊕)HF₂ ⁻, (C₈H₁₇)₄ P^(⊕)H₂F₃ ^(⊖), (Me₂N)₃P═N^(⊕)═P(NMe₂)₃F^(⊖), BuNMe₃ F^(⊖),

KF(HF)z₁, z₁=0, 1, 2, 3 or 4

CsF(HF)z₁, z₁=0, 1, 2, 3, or 4

NaF(HF)z₁, z₁=0, 1, 2, 3, or 4

NaF(HF)z₁, z₁=0, 1, 2, 3, or 4

polyvinyl pyridine(HF)z₃, z₃=1-7,

polyethylene —CH₂N^(⊕)Me₃ ^(⊖)F^(⊕),

Et₃NH^(⊕)(HF) z₄F^(⊖), wherein z₄=1-3, H₂SiF₆ and the like.

The ionic fluoride salts are either known compounds or are prepared byart recognized techniques. Their synthesis is exemplified by thesynthesis of benzyltriphenyl phosphonium dihydrogen-trifluoride. In thiscase, an excess of the fluoride salt, e.g., KHF₂, is reacted with a halosalt, (PQ₅Q₆Q₇Q₈ Hal, i.e., where P, Q₅, Q₆, Q₇ and Q₈ are defined aboveand Hal is Bromo, Chloro or Iodo) e.g., benzyltriphenylphosphoniumchloride. The reaction is effected in a solvent which will dissolve bothreagents and the product, such as water and the desired product isseparated therefrom. This reaction may be effected at temperatures abovethe freezing point of the solvent (e.g., water) and up to the boilingpoint thereof, but preferably it is performed at room temperature.

As used herein, the term “alkyl”, when used alone or in combination withother groups, refers to a carbon chain containing from one to twentycarbon atoms. It may be a straight chain or branched and includes suchgroups as methyl, ethyl, propyl, isopropyl, n-pentyl, amyl, hexyl,heptyl, octyl, nonyl, decyl, pentadecyl, hexadecyl, and the like. Thepreferred alkyl groups are lower alkyl groups containing 1-6 carbonatoms. It is even more preferred that alkyl contains from 1-3 carbonatoms. It is most preferably methyl.

The term “aryl” as used herein, alone or in combination, refers to anaromatic ring system containing from 6-10 ring carbon atoms and up to atotal of 15 carbon atoms. It includes such groups as phenyl, α-naphthyl,β-naphthyl, and the like.

“Aralkyl groups” are alkyl groups attached to the aryl group through analkylene bridge. Such groups include phenethyl, phenpropyl and mostpreferably benzyl.

“Cycloalkyl” as used herein refers to a cycloalkyl group containing onlyring carbon atoms and from 3-10 ring carbon atoms and up to a total of15 carbon atoms. It may consist of 1 ring or two fused rings or threefused rings. Examples include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, decalinyl, norbornyl,adamanty and the like.

“Cycloalkylalkyl” denotes alkyl groups attached to the cycloalkyl groupthrough an alkylene bridge. Such groups include cyclohexylmethyl,cyclopentylethyl, and the like.

Unless indicated to the contrary, halogen, as used herein refers tofluorine, chlorine, bromine or iodine.

As employed herein, the term “heteroaryl” is a heteroaromatic containingat least one heteroatom ring atom selected from nitrogen, sulfur andoxygen and up to a maximum of four ring heteroatoms. The heteroarylgroup contains from 5 to 14 ring atoms and up to a total of 13 ringcarbon atoms and a total of 18 carbon atoms. The heteroaryl group may bemonocyclic, bicyclic or tricyclic. Also included in this expression arethe benzoheteroaromatic.

The heteroaryl group preferably contains no more than two ringheteroatoms, and most preferably contains one ring heteroatom. The mostpreferred ring heteroatoms are oxygen and nitrogen, with nitrogen beingthe most preferred.

If nitrogen is a ring atom, N-oxides can also be formed. The presentinvention contemplates the N-oxides of the nitrogen containingheteroaryls.

Examples of heteroaryls include thienyl, benzothienyl, 1-naphthothienyl,thianthrenyl, furyl, benzofuryl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl,indazolyl, purinyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, carbolinyl, isothiazolyl,isoxazolyl and the like. It is preferred that the heteroaryl group ispyridyl, pyrrolyl, furyl, indolyl, quninolyl, isoquinolyl or benzofuryl.Especially preferred is pyridyl.

Alkyl carbonyl refers to an alkyl group attached to the main chainthrough a carbonyl. Similarly, aryl carbonyl refers to an aryl groupattached to the main chain through a carbonyl group.

As used herein, an “electron donating group” shall designate a groupthat will release or donate electrons more than hydrogen would if itoccupied the same position in the molecule. See J. March, AdvancedOrganic Chemistry, 3rd Ed., John Wiley & Sons p. 238 (1985). These typesof groups are well known in the art. Examples include lower alkylamino,diloweralkylamino, amino, halo, aryl, lower alkoxy, hydroxy, loweraralkoxy, aryloxy, mercapto, lower alkylthio, and the like. Thepreferred electron donating groups are amino, hydroxy, lower alkoxy,lower alkylamino and diloweralkylamino.

The presence of all three elements, i.e., the ionic fluoride salt, thecoupling agent, and the N-α-amino protected amino acid or acylatingderivative thereof are required for the in situ preparation of theN-α-amino protected amino acid fluoride. The reaction is effected bymixing the three elements in a suitable solvent in which the amino acidor acylating derivative thereof, the ionic fluoride salt and thecoupling agent and the resulting amino acid fluoride are soluble, or arepartially soluble. Examples of suitable solvents includedimethylformamide, methylene chloride (DCM), N,N-dimethylpyrrolidine(NMP), THF, ethyl ether, dioxane, and the like. DMF and DCM are thepreferred solvents. The reaction may be effected at temperatures rangingfrom about 0° C. to the reflux temperature of the solvent, but it ispreferred that the reaction be effected at about room temperature.

Effective amounts of the fluoride additive, the coupling agent and theN-α-amino protected amino acid or acylating derivative thereof are usedto form the N-α-amino protected amino acid fluoride in situ. Preferablythe ratio of ionic fluoride salt to amino acid ranges from about 1:5 toabout 5:1 equivalents, and more preferably from about 1:1 to about 5:1equivalents, respectively, and most preferably, the ratio is about 1:1equivalents. Similarly, the ratio of coupling agent to amino acid rangesfrom about 1:5 to about 5:1 equivalents, and more preferably from about1:1 to about 5:1, respectively and most preferably the ratio is about1:1. Finally, it is preferred that the ratio of ionic fluoride salt tocoupling agent ranges from about 1:10 to about 10:1 equivalents, andmore preferably from about 1:5 to about 5:1 equivalents, respectivelyand the ratio is most preferably about 1:1 equivalents. In fact, it ismost preferred that the equivalent ratio of amino acid: fluoride salt:coupling reagent is about 1:1:1.

The amino acid fluoride thus formed, in situ, is produced withoutracemization. In other words, the L amino acid would produce the L-aminoacid fluoride. Similarly, the D-amino acid fluoride would be preparedfrom the corresponding D-amino acid.

The amino acid fluoride thus formed can then react with an amino acidhaving a free α-amino group or peptide having a free α-amino group toproduce a protected peptide in accordance with standard techniques knownin the art. Removal of the protecting groups affords the desiredpeptide.

This process can be repeated to form tripeptides, tetrapeptides, orhigher peptides until the desired product is attained. The scope of thepresent process is broad, as the amino acid fluorides prepared inaccordance with the first step can be coupled with an amino acid,dipeptide, tripeptide, or higher peptide, as long as it has a freeα-amino group.

Inasmuch as the amino acid fluoride prepared in accordance with thepresent invention is formed in situ, it reacts immediately with thepeptide or amino acid having a free amino group already present in thereaction mixture. Thus, another aspect of the present invention is thepreparation of peptides. Effective amounts of the amino acid fluorideformed in situ is reacted with the amino acid or peptide having a freeα-amino group. Preferably, the equivalent ratio of amino acid fluorideto the amino acid peptide having a free amino group ranges from about1:1 to about 10:1, respectively, and most preferably from about 2:1 toabout 4:1.

The coupling reaction usually takes place in an inert organic solventsuch as dimethylformamide (DMF), methylene chloride (DCM),N-methylpyrrolidine (NMP), ethyl ether, THF, dioxane, or the like. Infact, DMF, or DCM is the preferred solvent in solid phase synthesisbecause of the favorable solvation properties of each. The reactiontakes place under mild conditions usually ranging from about 0° C. toabout 30° C. After the peptide is formed, the blocking groups areremoved by techniques known to one skilled in the art.

The process described herein with the formation of the N-α-aminoprotected amino acid fluoride in situ followed by the coupling reactiondescribed hereinabove, is applicable in both solution phase and solidphase synthesis. The synthesis of higher peptides according to thepresent invention is effected by constantly repeating the followingsequence of steps:

1) formation of an amino acid fluoride in situ by reacting theappropriate amino acid or acylating derivative thereof, a coupling agentand the ionic fluoride salt;

2) reacting the amino acid fluoride produced in situ with a second aminoacid having a free amino group and protected a carboxy group or peptidehaving a free amino group, and a protected carboxyl group to form apeptide bond; and

3) removal of the protecting groups By repeating steps 1-3, higherpeptides are formed. This is clearly shown by the following sequence:

In the above scheme, Ly, and z are as defined hereinabove, and AA₁, AA₂,AA₃, and AA₄ are independently N-α-amino protected amino acid. BLK is aα-amino blocking group, and P is a peptidyl-resin in solid phasesynthesis or a carboxy protecting group commonly used in solutionpeptide synthesis, such as the methyl ester, t-butylester,β-trimethylsilyethyl, benzyl ester, and the like. A variety of carboxyprotecting groups known in the art may be employed. Examples of many ofthese possible groups can be found in “Protective groups in OrganicSynthesis”, by T. W. Green, John Wiley and Sons, 1981, the contents ofwhich are incorporated by reference.

In the above sequence, the ionic fluoride salt was represented byLy(HF)zF^(⊖). However, this was just exemplary as the above sequence ofsteps could be effected if the anion of the ionic fluoride salt wereTG₁G₂G₃F₂ ⁻.

As shown by the above scheme, the N-α-amino protected amino acid isreacted with an ionic fluoride salt, and a coupling agent to form anN-α-amino protected amino acid fluoride in situ which is reacted with asecond amino acid in which the carboxy group is protected and which hasa free amino group. A peptide is formed between the first amino acid andthe second amino acid. The peptide chain can be increased by removingthe α-amino protecting group by techniques known to one skilled in theart. Another N-α-amino protected amino acid fluoride is formed in situin accordance with the present invention and this is 3reacted with thedipeptide formed hereinabove to produce the N-α-amino protectedtripeptide. The N-α-amino protecting group of the tripeptide is removedand the above cycle is repeated until the desired peptide has beenobtained. In the very last step the protecting groups, i.e., theN-α-amino protecting group, the α-carboxy protecting group and theprotecting groups on the side chains, if any, are removed.

The present invention can readily be utilized in solid phase peptidesynthesis. Solid phase peptide synthesis is based on the stepwiseassembly of a peptide chain while it is attached at one end to a solidsupport or solid phase peptide resin. Two methods are generally wellknown in the art.

One, the Merrifield method, employs a solid support for attachment ofthe amino acid or peptide residues. This method employs N-protectedamino acids as building blocks which are added to an amino acid orpeptide residue attached to the solid support at the acyl (acid) end ofthe molecule. After the peptide bond has been formed, the protectedgroup is removed and the cycle repeated. When a peptide having thedesired sequence has been synthesized, it is then removed from thesupport.

The second method, the inverse Merrifield method, employs reagentsattached to solid supports in a series of columns. The amino acid orpeptide residue is passed through these columns in a series to form thedesired amino acid sequence.

These methods are well known in the art as discussed in U.S. Pat. Nos.4,108,846, 3,839,396, 3,835,175, 4,508,657, 4,623,484, 4,575,541,4,581,167, 4,394,519 as well as in Advances in Enzymology, 32,221 (1961)and in PEPTIDES, Vol, 2, edited by Erhard Gross and JohannesMeienhoffer, Academic Press, New York, N.Y. pp. 3-255 (1980) and all ofthese are incorporated herein by reference as if fully set forth herein.

One of the surprising results accompanying the preparation of peptidesutilizing the in situ preparation of amino acid fluoride of the presentinvention is the higher yields of peptide product that is formed ascompared when the ionic fluoride salt is not present. Without wishing tobe bound, it is believed that this phenomenon can be explained bycomparing the mechanism of action in the absence and presence of ionicfluoride salt.

In the absence of ionic fluoride salt, the reaction between the couplingagent and the amino acid can proceed via several different pathways,some of which leads to by-products. This is illustrated in the followingscheme, wherein T₁N═C—NT₁ represents an exemplary coupling agent (e.g.,carbodiimides wherein T₁ may be alkyl or cycloalkyl), UCO₂H represents afirst amino acid with a free carboxy group and VNH₂ represents a secondamino acid with a free amino group:

The final product, amide 12, can be derived from the unstable O-acylurea7, its protonated form 9 or the symmetric anhydride 10 which is derivedfrom 9 by reaction with the acid anion. Formation of the rearrangementproduct 8 represents loss of active material since this reaction isirreversible. In the presence of fluoride ionic salt, activeintermediate 7 is diverted to give the acid fluoride, thus accountingfor the excellent results obtained in the work described here (SchemeII).

Under optimum conditions, all of the acid is converted to the acidfluoride, thus serving to guarantee an efficient coupling process as theacid fluoride reacts with the amino component of the amino acid. It isbelieved to be a direct process without the multiple pathway stepsbelieved to be present when the ionic fluoride salt is absent.Therefore, the process is more efficient in the presence of the ionicfluoride salt.

Moreover, by the in situ process of the present invention, amino acidfluorides can be prepared which heretofore, could not be prepared. Forexample, as shown hereinbelow, FMOC—His(Trt)—F can be prepared withoutsignificant racemization.

Another aspect of the present invention is directed to a kit comprisingthe three elements described hereinabove for forming the amino acidfluoride in situ. More specifically, this aspect of the invention isdirected to a kit comprising in a first compartment an N-α-aminoprotected amino acid or acylating derivative thereof, in a secondcompartment a coupling agent and in a third compartment an ionicfluoride salt as described herein. A variation thereof is a kitcomprising two compartments, wherein the first compartment contains theionic fluoride salt of the present invention admixed with the N-α-aminoprotected amino acid or acylating derivative thereof and a secondcompartment containing the coupling agent. Thus, when ready for use theamino acid, the coupling agent and the ionic fluoride salt are removedfrom each compartment and mixed together with an amino acid or peptidehaving a free amino group in the appropriate solvent, thereby generatinga peptide from the in situ formed N-α-amino protected amino acidfluoride.

Another variation is a kit comprising any two of the elements describedhereinabove. More specifically, a variation is directed to a kitcomprising in a first compartment the ionic fluoride salt as describedhereinabove and in a second compartment the N-α-amino protected aminoacid. Alternatively, the kit contains 1 compartment containing theN-α-amino protected amino acid admixed with the ionic fluoride salt.Alternatively, a kit is comprised of two compartments, one compartmentcontaining the ionic fluoride salt and the second compartment containingthe coupling agent.

Thus, when these latter kits are ready for use, the ionic fluoride saltand either the N-α-amino protected amino acid or coupling agent is mixedin solution containing the third element (i.e., the coupling agent orthe N-α-amino protected amino acid, respectively) and an amino acid orpeptide having a free amino group to generate the desired peptide.

It is to be noted that the in situ process described hereinabove is notlimited to the preparation of amino acid fluorides in situ. The couplingagent and the ionic fluoride salt can be reacted with an organiccarboxylic acid especially hydrocarbyl organic acids, to form thecorresponding organic carboxylic acid fluoride in situ.

Another aspect of the present invention is directed to novel ionicfluoride salts. The ionic fluoride salts described herein generally havethe formula:

Cat—Ani

wherein

Cat is an alkali metal cation, alkaline earth metal cation, hydrogencation, NH₄+, NQ₅Q₆Q₇Q₈ ^(⊕), PQ₅Q₆Q₇Q₈⊕, SQ₅Q₆Q₇⊕, Hal Q₅Q₆⊕ or

(Me₂)N₃ P═N═P—N(Me₂)₃ and

Ani is a fluoride salt anion of the formula:

Ly(HF)_(z)F^(⊖) or TG₁G₂G₃F₂ ^(⊖)

wherein Q₅, Q₆, Q₇, Q₈, Hal, Ly, z, T, G₁, G₂, G₃, G₄ are as definedherein.

It is preferred that Q₅ is arylalkyl and Q₆, Q₇ and Q₈ are aryl,especially phenyl.

It is to be understood, unless indicated to the contrary, that the aryl,aryl alkyl groups, alkyl groups, cycloalkyl group, cycloalkyl alkylgroups and the other groups defined herein may be unsubstituted orsubstituted with an electron donating group or electron withdrawinggroups, including alkyl groups, which do not interfere with the reactionfor forming the amino acid fluoride or the coupling reaction, describedherein.

The invention will now be illustrated by examples. The examples are notintended to be limiting of the scope of the present invention. Inconjunction with the general and detailed descriptions above, theexamples provide further understanding of the present invention:

EXAMPLE 1

Benzyltriphenylphosphonium Dihydrogentrifluoride

A solution of 7.8 g (20 mmol) benzyltriphenylphosphonium chloride in 100ml of CH₂Cl₂ was stirred vigorously with a solution of 78.2 g (1 mole)of KHF₂ in 200 ml of distilled water for 0.5 h in a plastic flask. Afterseparation of layers, the organic layer was stirred two more times asindicated above with fresh 200-mL portions of KHF₂ solution. Finally,the organic layer was separated and the solvent removed in vacuum. Thesolid residue upon recrystallization from methanol-ether gave acolorless crystalline mass (8.16 g, yield 99%), mp 148-150° C.; ¹H NMR(CDCl₃); δ 4.85 (d, 2, CH₂); 7.0-7.7 (m, 22); Anal. Calcd for C₂₅H₂₄PF₃;C, 72.80; H, 5.86; F, 13.82. Found: C, 72.95; H, 5.80; F, 13.60.

EXAMPLES 2-4

Manual Solid Phase Synthesis

In Examples 2-4, the following syntheses of peptides were utilized. Allsyntheses were carried out in 5- or 10-mL plastic syringes using aPAL-PEG-PS resin using either 4 or 5 eqs. excess amino acid, deblockingtimes of 7 min. and coupling times of 30 min. Preactivation via DCC orDIC was carried out in some cases in CH₂Cl₂ followed by evaporation ofsolvent and transfer to DMF for coupling. In other cases DMF was usedfor both activation and coupling. Parallel runs with and withoutadditive were carried out side-by-side.

For non-carbodiimide coupling reagents, e.g., TFFH, HATU and TBTU,parallel runs were made similarly in the absence and presence of ionicfluoride salt. Co-injection experiments confirmed that the presence offluoride salt always guaranteed the best result.

EXAMPLE 2

H—Tyr—Aib—Aib—Phe—Leu—NH₂

Using the above procedure, and various coupling agents in the presenceand absence of ionic fluoride salts, the above pentapeptide was preparedand the results compared. The data is tabulated in Table 1.

TABLE 1 H-Tyr-Aib-Aib-Phe-Leu-NH₂ ^(a) des- Coupling Proactivation ionicfluoride Yield Aib-4- Solvent Reagent Base^(b) Time, min. salt (%)mer(%) DMF HATU/HOAT DIEA(2) 7 — 25 60.5 DMF HATU DIEA(2) 7 — 83.4 4.5DMF TFFH DIEA(2) 7 — 87.1 9.0 CH₂Cl₂/ DCC DIEA(1) 7 — 50.6 58.8 DMF^(c)CH₂Cl₂/ DCC — 7 — 90.3 73.5 DMF^(c) CH₂Cl₂/ DCC — 7 (C₆H₅)₄PH₂F₃ 97.810.5 DMF^(c) DMF TFFH DIEA(2) 7 (C₆H₅)₄PH₂F₃ 71.2 7.1 DMF DCC — 7(C₆H₅)₄PH₂F₃ 60.4 55.4 DMF DIC — 15 — 32.2 78.1 DMF DIC — 15(C₆H₅)₄PH₂F₃ 67.9 18.8 DMF DIC — 15 TsOH 75.9 26.5 DMF DIC — 15nBu₄N(⁺)(C₆H₅)₃ 40.8 49.1 SnF₂(−) ^(a)Manual syntheses; a 5-fold excessof the Fmoc amino acid was used with a coupling time of 30 min (single).^(b)The number of equivalents per equivalent of acid is given inparenthesis. ^(c)In these cases the preactivation was carried out inCH₂Cl₂ and the solvent evaporated so that the coupling reaction wascarried out in DMF.

It should be noted that the pentapeptide 14

H—Tyr—Aib—Aib—Phe—Leu—NH₂   14

gives via manual solid phase synthesis using DCC alone mainly thecorresponding tetrapeptide 15

H—Tyr—Aib—Phe—Leu—NH₂   15

due to the extreme difficulty of the Aib-Aib coupling step. The ratio ofdesired-to undesired peptide is 0.36. In contrast, in the presence oftetraphenylphosphonium dihydrogen trifluoride (TPP⁽⁺⁾H₂F₃ ⁽⁻⁾) thedesired pentapeptide 14 is obtained in a yield of 97.8% with a purity ofcrude product of 90% (ratio of desired to undesired, 8.44). For thissynthesis, the standard method of preactivating in DCM (methylenechloride) solvent followed by evaporation and coupling in DMF solutionwas followed. Direct preactivation in DMF can be speeded up by thepresence of a catalytic amount of a strong sulfonic acid. For comparisondata on the synthesis of pentapeptide 14, see Table 1.

Infrared studies in CH₂Cl₂ and DMF which allowed one to follow formationof the acid fluoride, symmetric anhydride and oxazolone as well asconversion of the last named acid fluoride were made under variousconditions. As an α,α-disubstituted amino acid Fmoc—Aib—OH 16 is readilyconverted to an oxazolone 17 so that initially a mixture of acidfluoride, symmetric anhydride, and oxazolone is formed (eq.5).

Depending on the conditions 17 is converted more or less readily to acidfluoride 18. Model studies showed that conversion of anhydride 19 toacid fluoride was slow and incomplete so that for practical purposes itis important that the conditions be such that anhydride is by-passed.This result is achieved by having the fluoride ionic salt present fromthe start of the reaction. The speed and extent of formation of 18determines the overall ease of peptide bond formation.

EXAMPLE 3

Using the methodology indicated hereinabove, the following peptides wereprepared:

H—Val—Gln—Ala—Ala—Ile—Asp—Tyr—Ile—Asn—Gly—NH₂   20

Ac—Aib—Pro—Aib—Ala—Aib—Ala—Gln—Aib—Val—Aib—Gly—Leu—Aib—Pro—Val—Aib—Aib—Glu—Gln—Phe—NH₂  21

The results showed that for ACP (65-74) 20 a manual solid phasesynthesis using DIC (preactivation for 7 min. in CH₂Cl₂, evaporation ofthe solvent and dissolution of the residue in DMF) gave an excellentcrude product. If base (DIEA) was added to the coupling step the crudepeptide was of lesser quality. With eight Aib residues and one Aib—Aibunit, peptide 21 represents a difficult challenge for solid phaseassembly. Previously only acid fluorides were successful in the solidphase synthesis of this model. Now the present method is also shown tobe successful.

EXAMPLE 4

Syntheses were also carried out on the ACP decapeptide which had beenmodified by substituting α-methylalanine (Aib) for the two alanineunits. As a general model this peptide 22 is quite

H—Val—Gln—Aib—Aib—Ile—Asp—Tyr—Ile—Asn Gly—NH₂   22

demanding yet is only half the size of alamethicin amide 21, allowingtwo test syntheses to be completed during the same time period.

With this new model available a number of other coupling systems havebeen examined in order to extend the generality of the ionic fluoridesalt. For example, TBTU by itself provided only a small amount of thedesired decapeptide 22; the main product being the des-Aib analog. Onthe other hand, TBTU in the presence of BTPPH₂F₃ gave peptide 22 as theonly major product. Similarly while results with HATU were better thanwith TBTU since a 50—50 mixture of the desired peptide and the des-Aibpeptide was obtained, the use of HATU along with the ionic fluoride saltgave only the desired peptide. It is thus to be emphasized that use ofTBTU with ionic fluoride salt, in accordance with the present invention,can equal or better the results obtained via HATU.

In the case of TFFH, results with and without ionic fluoride salt werenearly the same in terms of peptide quality, although the yieldincreased from 52% to 81% when the ionic fluoride salt was used. Otherinexpensive coupling reagents can be similarly modified to bring theircoupling level up to that of acid fluorides which are currently the mostefficient of the common coupling species. This includes the use of EDC,active esters of various kinds, mixed anhydrides, EEDQ, NCAs,(N-carboxyanhydrides), ethoxyacetylene, yneamines, ketene imines, andthe like.

The following experiments clearly further illustrate that the presenceof the ionic fluoride salts of the present invention provide aprotective a effect against racemization.

EXAMPLE 5

FMOC—Leu—Pro—NH₂

1. Flowsheet.

2. Conditions.

Fmoc—Pro—PAL—PEG—PS (0.2 mmol/g), 100 mg.

* Fmoc—Leu—OH, 3 eqs. excess, 0.06 mmol, 21.2 mg.

* Base, 6 eqs. excess-see Table 2.

* CR, 3 eqs. excess-see Table 2.

* Fluoride salt, 3 eqs. excess, 24.7 mg-see Table 2.

* Solvent: DMF (0.4 mL). Coupling time: 30 min.

*The asterisk identifies the components of the preactivation solution.For preactivation times see Table 2.

3. General Procedure.

In 5-mL syringe tubes fitted at the bottom with a Teflon frit wasweighed 100 mg of Fmoc—Pro—PAL—PEG—PS (0.2 mmol/g). The resin was washedwith DMF (5×5 mL), treated with 20% piperidine in DMF (4 mL) for 7 minand then washed with DMF (5×5 mL). After removing the solvent by wateraspirator vacuum the preactivated solution (see above and Table 2) wasadded to the resin. During the 30 min coupling time the resin wasstirred from time-to-time with a Teflon stick. At the end of 30 min theresin was washed with DMF (5×5 mL), CH₂Cl₂ (5×5 mL), EtOH (1×5 mL),ether (2×5 mL) and dried in vacuo for 0.5 h. The dry resin was treatedwith 2 mL of 100% trifluoroacetic acid (TFA) for 1 h. The TFA wasremoved by filtration into a collection vessel and the resin washed onthe filter with CH₂Cl₂ (2×5 mL). The combined filtrates wereconcentrated in vacuo at room temperature to dryness and the residuedissolved in 1 mL of CH₃CN for analysis by direct injection onto an HPLCcolumn.

4. HPLC Separation.

Column: Nova Pak, 4μ, C₁₈.

Solvent system: isocratic, 40% CH₃CN (0.1%

TFA)—60% H₂O (0.1% TFA).

Flow rate: 1 mL/min.

Detector: PDA at 220 nm.

The results are given in Table 2.

TABLE 2 System: Fmoc-Leu-OH + H-Pro-PAL-PEG-PS, with or without theUniversal Fluoride Salt (C₆H₅CH₂P(C₆H₅)₃ ^(⊕)H₂F₃ ^(⊖)) Preactiv.CR(amt.) Base(amt.) Additive time(min) DL(%) HATU(22.8 mg) DIEA(20.9 μL)− 1 0.96 HATU(22.8 mg) DIEA(20.9 μL) + 1 0.81 HATU(22.8 mg) DIEA(20.9μL) − 7 0.81 HATU(22.8 mg) DIEA(20.0 μL) + 7 0.82 HATU(22.8 mg)TMP**(15.9 μL) − 7 0.22 HATU(22.8 mg) TMP(15.9 μL) + 7 0.33 TBTU(19.2mg) DIEA(20.9 μL) − 1 0.82 TBTU(19.2 mg) DIEA(20.9 μL) + 1 0.79TBTU(19.2 mg) DIEA(20.9 μL) − 7 0.85 TBTU(19.2 mg) DIEA(20.9 μL) + 70.76 TBTU(19.2 mg) TMP(15.9 μL) − 7 0.26 TBTU(19.2 mg) TMP(15.9 μL) + 70.24 TFFH(15.9 mg) DIEA(20.9 μL) − 7 0.78 TFFH(15.9 mg) DIEA(20.9 μL) +7 0.74 TFFH(15.9 mg) DB(DMAP)*(28.3 mg) − 7 0.23 TFFH(15.9 mg)DB(DMAP)*(28.3 mg) + 7 0.25 *DB(DMAP) = 2,6-di-t-butyl-4-(dimethylamino)pyridine **TMP = trimethylpyridine (collidine)

EXAMPLE 6

Z—Phe—Val—Pro—NH₂

The same method as in Example 5 was utilized except that a firstcoupling (Fmoc—Val OH) was followed by a second (Z—Phe—OH). For theresults see Table 3.

TABLE 3 Assembly of Z-Phe-Val-Pro-NH₂ from H-Pro-PAL-PEG-PS, with orwithout the Universal Ionic Fluoride (C₆H₅CH₂P(C₆H₅)₃ ^(⊕)H₂F₃ ^(⊖))Preactiv. CR(amt.) Base(amt.) Additive time(min.) D-Fhe D-Val HATU(45.6mg) DIEA(41.8 μl) − 7 0.81 <0.1 TFFH(31.7 mg) DIEA(41.8 μl) − 7 0.180.43 TFFH(31.7 mg) DIEA(41.8 μl) + 7 0.19 <0.1

Heretofore, the coupling of histidine was always considered difficultwithout significant racemization. However, as clearly shown by thefollowing data, the use of the ionic fluoride provides a protectiveeffect.

EXAMPLE 7

Fmoc—His(Trt)—Pro—NH₂

The same method was applied except that a single coupling viaFmoc—His(Trt)—OH was examined. The HPLC conditions were the same exceptfor use of an isocratic system made of 24% CH₃CN (0.1% TFA)—76%H₂O (0.1%TFA). For the results, see Table 4.

TABLE 4 System: Fmoc-His(Trt)-OH + H-Pro-PAL-PEG-PS, with or without theUniversal Fluoride Additive (C₆H₅CH₂P(C₆H₅)₃ ^(⊕)H₂F₃ ^(⊖)) Preactiv.CR(amt.) Base(amt.) Additive time(min) DL(%) TFFH(31.7 mg) DIEA(41.8 μL)− 7 7.40 TFFH(31.7 mg) DIEA(41.8 μL) + 7 1.82 TBTU(38.5 mg) DIEA(41.8μL) − 7 2.79 TBTU(38.5 mg) DIEA(41.8 μL) + 7 1.45 HATU(45.6 mg)DIEA(41.8 μL) − 7 3.24 HATU(45.6 mg) DIEA(41.8 μL) + 7 1.52

The protective effective provided by the ionic fluoride salt is mostclearly shown in the case of the very sensitive histidine case (Table4). For TFFH the racemization dropped from 7.4 to 1.8% when additive waspresent. For the base used (DIEA) the values for all three couplingreagents were similar suggesting that the same intermediate is involved.It seems likely that the presence of the complexed protonic species(H₂F₃ ^(⊖)) maintains an appropriate pH for the reaction and avoids theeffect seen with the strong base diisopropylethylamine present alone.Similar effects occur for other sensitive amino acids (α-phenylglycine,cysteine, etc.).

The above preferred embodiments and examples are given to illustrate thescope and spirit of the present invention. These embodiments andexamples will make apparent to those skilled in the art otherembodiments and examples. These other embodiments and examples arewithin the contemplation of the present invention. Therefore, thepresent invention should be limited only by the appended claims.

What is claimed is:
 1. An in situ process for preparing a N-∝-protectedamino acid fluoride which comprises mixing a N-∝ protected amino acid oracylating derivative thereof with a fluorinating effective amount of anionic fluoride salt in the presence of a coupling agent under conditionseffective to form said protected amino acid fluoride, wherein the anionof the ionic fluoride salt has the formula: Ly (HF)₂F^(⊖), TG₁G₂G₃F₂— orLF^(⊖); wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; y is 0 or 1; L isTG₁G₂G₃G₄; T is a group IV element selected from the group consisting ofSi, Ge, Sn and Pb; and G₁, G₂, G₃ and G₄ are independently halogen,hydrogen,, alkyl, aryl, arylalkyl, cycloalkyl, or cycloalkylalkyl. 2.The process according to claim 1 wherein the anion of the fluoride saltis (HF)zF^(⊖) wherein z is 1-10.
 3. The process according to claim 1wherein G₁, G₂, G₃ and G₄ are independently alkyl, arylalkyl or aryl. 4.The process according to claim 3 wherein alkyl contains 1-6 carbonatoms.
 5. The process accordingly claim 1 wherein z is 1, 2 or
 3. 6. Theprocess according to claim 1 wherein the coupling agent is acarbodiimide.
 7. The process according to claim 6 wherein thecarbodiimide is DCC, DIC, or EDC.
 8. The process according to claim 1wherein the coupling agent has the formula:

or N-oxides thereof or salt thereof wherein R₁ and R₂ taken togetherwith the carbon atoms to which they are attached form a heteroaryl ringor aryl ring wherein said heteroaryl ring is an oxygen, sulfur ornitrogen containing heteroaromatic having from 3 and up to a total of 13ring carbon atoms, said heteroaryl or aryl may be unsubstituted orsubstituted with lower alkyl or an electron donating group; Y is O, NR₄,CR₄R₅; R₄ and R₅ are independently hydrogen or lower alkyl; X is CR₆R₇or NR₆; R₆ and R₇ are independently hydrogen or lower alkyl or R₆ and R₇taken together form an oxo group or when Q is R₄ and R₆ taken togethermay form a bond between the nitrogen or carbon atom of Y and thenitrogen or carbon atom of X; Q is CR₈R₉ or NR₈; R₈ and R₉ areindependently hydrogen or lower alkyl or R₄ and R₈ taken together mayform a bond between the ring carbon or nitrogen atom of Q and the ringcarbon or nitrogen atom of R₈; and R₃ is hydrogen, lower alkyl carbonyl,aryl carbonyl, lower aryl alkyl carbonyl, a positively charged electronwithdrawing group, or SO₂R₁₄; and R₁₄ is hydrogen or lower alkyl.
 9. Theprocess according to claim 8 wherein R₃ is

wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently hydrogen, alkyl, orlower alkoxy, lower alkyl or R₁₀ and R₁₂ taken together with the atomsto which they are attached form a ring so that R₃ become

wherein m₂ is 0 or 1, or R₁₀ and R₁₁ taken together with the nitrogen towhich they are attached form a 5- or 6-membered heterocyclic ringcontaining up to 5 carbon ring atoms.
 10. The process according to claim8 wherein the coupling agent is of the formula

wherein A is N or CR₁₅; D is CR₁₆ or N; E is CR₁₇ or N; G is CR₁₈ or N;R₁₅, R₁₆, R₁₇ and R₁₈ are independently hydrogen, lower alkyl orelectron donating group; J is NR₁₅, O, CR₁₅R₁₉ or S(O)p; p is 0, 1 or 2,and R₁₉ is hydrogen or lower alkyl.
 11. The process according to claim10 wherein the coupling agent is of the formula:


12. The process according to claim 11 wherein R₃ is lower alkylcarbonyl,


13. The process according to claim 1 wherein the anion of the ionicfluoride is HF₂ ^(⊖), H₂F₃ ^(⊖), Snφ₃F₂ ^(⊖), or H₃F₄ ^(⊖), wherein φ isphenyl.
 14. The process according to claim 1 wherein the coupling agentis selected from the group consisting of HBTU, HATU, TBTU, TFFH, DCC,EEDQ and DIC.
 15. The process according to claim 1 wherein the anion ofthe ionic fluoride salt has the formula: Ly(HF)_(z) F⁻ wherein z is 1,2, 3, 4, 5, 6, 7, 8, 9, or 10; y is 0 or 1; L is TG₁G₂G₃G₄; T is a GroupIV element selected from the group consisting of Si, Ge, Sn and Pb; andG₁, G₂, G₃ and G₄ are independently halogen, hydrogen, alkyl, aryl,arylalkyl, cycloalkyl or cycloalkyl alkyl.
 16. The process according toclaim 15 wherein the anion has the formula HF₂ ⁻, H₂F₃ ⁻, or H₃F₄ ⁻. 17.The process according to claim 15 wherein y is
 1. 18. The processaccording to claim 17 wherein z is 1, 2 or
 3. 19. The process accordingto claim 18 wherein G₁, G₂, G₃ or G₄ are independently alkyl, arylalkylor aryl.
 20. The process according to claim 17 wherein T is Sn or Si.21. The process according to claim 17 wherein G₁, G₂, G₃ and G₄ areindependently alkyl, arylalkyl or aryl.
 22. The process according toclaim 1 wherein the cation is an alkali metal, alkaline earth metal,hydrogen, ammonium, NQ₅Q₆Q₇Q₈ ⁺, PQ₅Q₆Q₇Q₈ ⁺, SQ₅Q₆Q₇ ⁺, HalQ₅Q₆ ^(⊕),or

wherein Q₅, Q₆, Q₇ and Q₈ are independently hydrogen, halogen, loweralkyl, aryl or aryl lower alkyl, and Hal is halo.
 23. The processaccording to claim 1 wherein the ionic fluoride salt has the formula:

wherein z is 1-6.
 24. The process according to claim 1 wherein the ionicsalt has the formula Et₃NH⁺(HF)z₄ F⁻, wherein z₄ is 1-3.
 25. The processaccording to claim 22 wherein y is
 0. 26. The process according to claim22 wherein y is
 1. 27. The process according to claim 26 wherein T is Snor Si.
 28. The process according to claim 26 wherein G₁, G₂, G₃ and G₄are independently alkyl, arylalkyl or aryl.
 29. The process according toclaim 22 wherein z is 1-4.
 30. The process according to claim 22 whereinthe cation is alkali, N^(⊕)Q₅Q₆Q₇Q₈ or P^(⊕)Q₅Q₆Q₇Q₈.
 31. The processaccording to claim 1 wherein the ionic fluoride salt is (C₆H₅)₄P^(⊕)H₂F₃^(⊖), benzyltriphenylphosphonium dihydrogen trifluoride, Bu₄N^(⊕)HF₂^(⊖), Bu₄P⁺HF₂ ⁻, Bu₄P⁺H₂F₃ ⁻, (C₆H₅)₄P^(⊕)HF₂ ⁻, (C₆H₅)₄P⁺H₂F³⁻,(C₈H₁₇)₄N⁺HF₂ ⁻, or (C₈H₁₇)₄P⁺H₂F₃ ⁻.
 32. The process according to claim31 wherein the ionic salt is benzyltriphenyl phosphonium dihydrogentrifluoride or (C₆H₅)₄P^(⊕)H₂F^(⊖) ₃.
 33. The process according to claim1 wherein the anion of the ionic fluoride salt has the formula TG₁G₂G₃F₂⁻ wherein T is a group IV element selected from the group consisting ofSi, Ge, Sn and Pb; G₁, G₂ and G₃ are independently halogen, hydrogen,alkyl, aryl, arylalkyl, cycloalkyl or cycloalkyl.
 34. The processaccording to claim 33 wherein T is Sn or Si.
 35. The process accordingto claim 33 wherein G₁, G₂ and G₃ and are independently alkyl, aryl, orarylalkyl.
 36. The process according to claim 34 wherein G₁, G₂, G₃ andG₄ are independently alkyl, aryl or arylalkyl.
 37. The process accordingto claim 1 wherein the anion of the ionic fluoride salt has the formula:TG₁G₂G₃F₂ ⁻ and the cation is an alkali metal, alkali earth metal,hydrogen, ammonium, NQ₅Q₆Q₇Q₈₊, PQ₅Q₆Q₇Q₈ ⁺, SQ₅Q₆Q₆ ⁺, HalQ₅Q₆ ^(⊕) or(Me₂N)₃—P══N═P—(NMe₂)₃, wherein T is a Group IV element selected fromthe group consisting of Si, Ge, Sn and Pb; G₁, G₂ and G₃ areindependently halogen, hydrogen, alkyl, aryl, arylalkyl, cycloalkyl orcycloalkyl alkyl; and Q₅, Q₆, Q₇ and Q₈ are independently hydrogen,lower alkyl, aryl or aryl lower alkyl and Hal is halo.
 38. The processaccording to claim 37 wherein T is Sn or Si.
 39. The process accordingto claim 37 wherein G₁, G₂, G₃, and G₄ are independently alkyl or arylalkyl or aryl.
 40. The process according to claim 37 wherein the ionicfluoride salt has the formula: n—Bu₄N⁺(C₆H₅)₃SnF₂ ^(⊖).
 41. A processfor forming a peptide between a N-∝-amino protected amino acid oracylating derivative thereof and a second amino acid with a free aminogroup or a peptide containing a free amino group, which processcomprises: (a) mixing in situ said N-∝-amino protected amino acid with afluorinating effective amount of an ionic fluoride salt in the presenceof a coupling agent under conditions effective to form a N-∝-aminoprotected amino acid fluoride, and (b) reacting the product of (a) withsaid second amino acid or peptide under peptide forming conditions,wherein the anion of the ionic fluoride salt has the formula: Ly(HF)_(z)F⁻, TG₁G₂G₃F₂ ⁻or LF^(⊖); wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9or 10; y is 0 or 1; L is TG₁G₂G₃G₄; T is a group IV element selectedfrom the group consisting of Si, Ge, Sn and Pb; and G₁, G₂, G₃ G₄ areindependently halogen, hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, orcycloalkylalkyl.
 42. The process according to claim 41 wherein G₁, G₂,G₃ and G₄ are independently alkyl, arylalkyl or aryl.
 43. The processaccording to claim 42 wherein alkyl contains 1-6 carbon atoms.
 44. Theprocess according to claim 41 wherein z is 1, 2, or
 3. 45. The processaccording to claim 41 wherein the coupling agent is a carbodiimide. 46.The process according to claim 45 wherein the carbodiimide is DCC, DIC,or EDC.
 47. The process according to claim 41 wherein the coupling agenthas the formula:

or N-oxides thereof or salt thereof wherein R₁ and R₂ taken togetherwith the carbon atoms to which they are attached form a heteroaryl ringor aryl ring wherein said heteroaryl ring is an oxygen, sulfur ornitrogen containing heteroaromatic having from 3 and up to a total of 13ring carbon atoms, said heteroaryl or aryl may be unsubstituted orsubstituted with lower alkyl or an electron donating group; Y is O, NR₄,CR₄R₅; R₄ and R₅ are independently hydrogen or lower alkyl; X is CR₆R₇or NR₆; R₆ and R₇ are independently hydrogen or lower alkyl or R₆ and R₇taken together form an oxo group or hen Q is not present, R₄ and R₆taken together may form a bond between the nitrogen or carbon atom of Yand the nitrogen or carbon atom of X; Q is CR₈R₉ or NR₈; R₈ and R₉ areindependently hydrogen or lower alkyl or R₄ and R₈ taken together mayform a bond between the ring carbon or nitrogen atom of Q and the ringcarbon or nitrogen atom of R₈; and R₃ is lower alkyl carbonyl, arylcarbonyl, lower aryl alkyl carbonyl, a positively charged electronwithdrawing group, or SO₂R₁₄ and R₁₄ is hydrogen or lower alkyl.
 48. Theprocess according to claim 47 wherein R₃ is

wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently hydrogen, alkyl, orlower alkoxy lower alkyl or R₁₀ and R₁₂ taken together with the atoms towhich they are attached form a ring so that R₃ become

wherein m₂ is 0 or 1 or R₁₀ and R₁₁ taken together with the nitrogenatom to which they are attached form a 5- or 6-membered heterocyclicring containing up to 5 ring carbon atoms.
 49. The process according toclaim 47 wherein the coupling agent is of the formula

wherein A is N or CR₁₅; D is CR₁₆ or N; E is CR₁₇ or N; G is CR₁₈ or N;R₁₅, R₁₆, R₁₇ and R₁₈ are independently hydrogen lower alkyl or electrondonating group; J is NR₁₅, O, CR₁₅R₁₉ or S(O)p; p is 0, 1 or 2, and R₁₉is hydrogen or lower alkyl.
 50. The process according to claim 49wherein the coupling agent is of the formula:


51. The process according to claim 49 wherein R₃ is lower alkyl,


52. The process according to claim 41 wherein the anion of the ionicfluoride is HF₂ ^(⊖), H₂F₃ ^(⊖), Snφ₃F₂ ^(⊖) or H₃F₂ ⁻, wherein φ isphenyl.
 53. The process according to claim 41 wherein the coupling agentis selected from the group consisting of HBTU, HATU, TBTU, TFFH, DCC,EEDQ, EDC and DIC.
 54. In the improved process for forming an organicacid fluoride in situ, from an organic acid, the improvement comprisingreacting said organic acid with a fluorinating effective amount of anionic fluoride salt in the presence of a peptide coupling agent, underconditions effective to form said organic acid fluoride, wherein theanion of the ionic fluoride salt has the formula: Ly (HF)_(z)F⁻,TG₁G₂G₃F₂ ⁻ or LF^(⊖), wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; yis 0 or 1; L is TG₁G₂G₃G₄; T is a group IV element selected from thegroup consisting of Si, Ge, Sn and Pb; and G₁, G₂, G₃ G₄ areindependently halogen, hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, orcycloalkylalkyl.
 55. The improved process according to claim 54 whereinthe organic acid is a hydrocarbyl organic acid.
 56. In the synthesis ofa peptide wherein a first N-∝-amino protected amino acid is covalentlycoupled to a solid phase peptide synthesis resin, the N-∝-aminoprotecting group is cleaved off and the resulting free amino group isreacted under amide forming conditions with a N-∝-amino protected aminoacid fluoride, the resulting N-∝-amino protecting group is cleaved offand coupled to another protected amino acid fluoride and the cyclerepeated until the desired peptide has been obtained, and then thepeptide is cleaved from said resin, the improvement comprising preparingsaid amino acid fluoride in situ by reacting a N-∝-amino protected aminoacid or acylating derivative thereof with a fluorinating effectiveamount of an ionic fluoride salt in the presence of a coupling agent,under conditions effective to form said protected amino acid fluoride,wherein the anion of the ionic fluoride salt has the formula:Ly(HF)₂Fe^(⊖), LF^(⊖) or TG₁G₂G₃F₂ ⁻; wherein z is 1, 2, 3, 4, 5, 6, 7,8, 9 or 10; y is 0 or 1; L is TG₁G₂G₃G₄; G₁, G₂, G₃ and G₄ areindependently halogen, hydrogen, alkyl, aryl, aryl alkyl, cycloalkyl orcycloalkylalkyl; and T is a Group IV element selected from the groupconsisting of Si, Ge, Sn, and Pb.
 57. The improved process according toclaim 56, wherein the anion of the ionic fluoride salt is (HF)_(z)F^(⊖),wherein z is 1-10.
 58. In a process for forming an amide from thereaction of an organic amine and a carboxylic acid fluoride, theimprovement comprising forming the carboxylic acid fluoride in situ byreacting a carboxylic acid or acylating derivative thereof with afluorinating effective amount of an ionic fluoride salt in the presenceof a peptide coupling agent under conditions effective to form saidcarboxylic acid fluoride, which is then reacted with the organic amineto form said amide, wherein the anion of the fluoride salt has theformula: Ly(HF)_(z)F^(⊖), TG₁G₂G₃G₄F₂ ⁻ or LF⁻; wherein z is 1-10; y is0 or 1; L is TG₁G₂G₃G₄; T is a Group IV element selected from the groupconsisting of Si, Ge, Sn and Pb; and G₁, G₂, G₃ and G₄ are independentlyhalogen, hydrogen alkyl, aryl, arylalkyl, cycloalkyl or cycloalkylalkyl.59. The process according to claim 58 wherein the anion of the ionicfluoride salt is (HF)_(z)F^(⊕), wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9or
 10. 60. A solution comprising a fluorinating effective amount of anionic fluoride salt having an ionizable fluoride, a coupling agent andan N-∝-amino protected amino acid or acylating derivative thereof,wherein the anion of the ionic fluoride salt has the formula:Ly(HF)_(z)F⁻, TG₁G₂G₃F₂ ⁻ or LF^(⊖); wherein z is 1, 2, 3, 4, 5, 6, 7,8, 9 or 10; y is 0 or 1; L is TG₁G₂G₃G₄; and T is a Group IV elementselected from the group consisting of Si, Ge, Sn and Pb; and G₁, G₂, G₃and G₄ are independently hydrogen, alkyl, aryl, arylalkyl, cycloalkyl orcycloalkylalkyl, wherein the fluoride salt, coupling agent and Nocaminoprotected amino acid or acylating derivative are capable of forming aN-∝-amino acid fluoride in situ.
 61. The solution according to claim 60wherein the anion of the ionic fluoride salt is (HF)_(z)F^(⊖), wherein zis 1-10.
 62. The solution according to claim 60 wherein z is 1, 2, 3 or4.
 63. The solution according to claim 60 wherein the coupling agent isa carbodimide.
 64. The solution according to claim 63 wherein thecarbodimide is DCC, DIC or EDC.
 65. The solution according to claim 60wherein the coupling agent has the formula

or N-oxides thereof or salt thereof wherein R₁ and R₂ taken togetherwith the carbon atoms to which they are attached form a heteroaryl ringor aryl ring wherein said heteroaryl ring is an oxygen, sulfur ornitrogen containing heteroaromatic having from 3 and up to a total of 13ring carbon atoms, said heteroaryl or aryl may be unsubstituted orsubstituted with lower alkyl or an electron donating group; Y is O, NR₄,CR₄R₅; R₄ and R₅ are independently hydrogen or lower alkyl; X is CR₆R₇or NR₆; R₆ and R₇ are independently hydrogen or lower alkyl or R₆ and R₇taken together form an oxo group or when Q is not present, R₄ and R₆taken together may form a bond between the nitrogen or carbon atom of Yand the nitrogen or carbon atom of X; Q is CR₈R₉ or NR₈; R₈ and R₉ areindependently hydrogen or lower alkyl or R₄ and R₈ taken together mayform a bond between the ring carbon or nitrogen atom of Q and the ringcarbon or nitrogen atom of R₈; and R₃ is lower alkyl carbonyl, arylcarbonyl, lower aryl alkyl carbonyl, a positively charged electronwithdrawing group, or SO₂R₁₄; and R₁₄ is hydrogen or lower alkyl. 66.The solution according to claim 60 wherein the anion of the ionicfluoride is HF₂ ⁻, H₂F₃ ⁻, H₃F₄ ⁻ or Snφ₃F₂ ⁻, wherein φ is phenyl. 67.The solution according to claim 60 wherein the ionic fluoride salt isbenzyl triphenylphosphonoim dihydrogen trifluoride.
 68. The solutionaccording to claim 60 wherein a α-carboxy protected amino acid having afree α-amino group is additionally present.
 69. The solution accordingto claim 60 wherein a peptide having a free-α-amino group isadditionally present.
 70. The solution according to claim 60 wherein theanion of the fluoride salt has the formula: Ly(HF)_(z) F⁻ wherein z is1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; y is 0 or 1; L is TG₁G₂G₃G₄; T is aGroup IV element selected from the group consisting of Si, Ge, Sn andPb; and G₁, G₂, G₃ and G₄ are independently halogen, hydrogen, alkyl,aryl, enyl alkyl, cycloalkyl or cycloalkyl.
 71. The solution accordingto claim 70 wherein y is
 0. 72. The solution according to claim 70wherein y is
 1. 73. The solution according to claim 72 wherein G₁, G₂,G₃ and G₄ are independently, alkyl, arylalkyl or aryl.
 74. The solutionaccording to claim 72 wherein T is Sn or Si.
 75. The solution accordingto claim 70 wherein the anion of the ionic fluoride salt has theformula: TG₁G₂G₃F₂ ⁻.
 76. The solution according to claim 75 wherein Tis Sn or Si.
 77. The solution according to claim 75 wherein G₁, G₂ andG₃ are independently alkyl, aryl or arylalkyl.
 78. The solutionaccording to claim 60 wherein the ionic fluoride salt has the formula

wherein z is 1-6.
 79. The solution according to claim 60 wherein theionic fluoride salt has the formula Et₃NH⁺(HF)z₄F⁻, wherein z₄ is 1-3.80. The solution according to claim 60 wherein the ionic fluoride saltis (C₆H₅)₄P^(⊕)H₂F₃ ^(⊖), benzyltriphenylphosphonium dihydrogentrifluoride, Bu₄N^(⊕)HF₂ ^(⊖), Bu₄P⁺HF₂ ⁻, Bu₄P⁺H₂F₃ ⁻, (C₆H₅)₄P^(⊕)HF₂⁻, (C₆H₅)₄P⁺H₂F₃ ⁻, (C₈H₁₇)₄N⁺HF₂ ⁻, or (C₈H₁₇)₄P⁺H₂F₃ ⁻.
 81. Thesolution according to claim 60 wherein the ionic fluoride salt is(C₆H₅)₃ (C₆H₅CH₂)P^(⊕)H₂F⁻ ₃, (C₆H₅)₄P^(⊕)H₂F⁻ ₃ or n—Bu₄N⁺(C₆H₅)₃SnF₂⁻.
 82. A kit useful for preparing amino acid fluorides in situcomprising an ionic fluoride salt and an N-∝-amino protected amino acid,the contents of said kit, when mixed with a coupling agent in solution,generates an amino acid fluoride in situ, the anion of the ionicfluoride salt having the formula: Ly(HF)₂F⁻, TG₁G₂G₃F₂ ⁻ or LF^(⊖);wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; y is 0 or 1; L isTG₁G₂G₃G₄; T is a Group IV element selected from the group consisting ofSi, Ge, Sn and Pb; and G₁, G₂, G₃ and G₄ are independently halogen,hydrogen, alkyl, aryl, arylalkyl, cycloalkyl or cycloalkylalkyl.
 83. Akit useful for preparing amino acid fluorides in situ comprising anionic fluoride salt in one compartment and a coupling agent in adifferent compartment, the contents of said kit when mixed with anN-∝-amino-protected amino acid or acylating derivative in solutiongenerates an N-∝-amino protected-amino acid fluoride in situ, the anionof the ionic fluoride salt having the formula: Ly(HF)₂F⁻, TG₁G₂G₃F₂ ⁻ orLF^(⊖); wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; v is 0 or 1 ; L isTG₁G₂G₃G₄; T is a Group IV element selected from the group consisting ofSi, Ge, Sn and Pb; and G₁, G₂, G₃ and G₄ are independently halogen,hydrogen, alkyl, aryl, arylalkyl, cycloalkyl or cycloalkylalkyl.
 84. Thekit according to claim 82 wherein the ionic fluoride salt is admixedwith the N-α-amino protected amino acid.
 85. The kit according to claim82 wherein the ionic fluoride salt is in one compartment and theN-α-amino protected amino acid is in a second compartment.
 86. The kitaccording to claim 84 wherein the coupling agent is additionally presentin a second compartment.
 87. The kit according to claim 85 wherein thecoupling agent is additionally present in a third compartment.
 88. Thekit according to claim 82, wherein the anion of the ionic fluoride saltis (HF)_(z)F^(⊖), wherein z is 1-10.
 89. A process for forming an aminoacid fluoride in situ comprising reacting a fluorinating effectiveamount of an ionic fluoride salt with a mixed anhydride of a N-∝-aminoacid, under conditions effective to form said amino acid fluoride,wherein the anion of the ionic fluoride salt has the formula:Ly(HF)_(z)F⁻, TG₁G₂G₃F₂ ⁻ or LF^(⊖); wherein z is 1, 2, 3, 4, 5, 6, 7,8, 9 or 10; y is 0 or 1; L is TG₁G₂G₃G₄; T is a Group IV elementselected from the group consisting of Si, Ge, Sn and Pb; and G₁, G₂, G₃and G₄ are independently halogen, hydrogen, alkyl, aryl, arylalkyl,cycloalkyl or cycloalkylalkyl.
 90. The process according to claim 89wherein the mixed anhydride is a lower carbonic acid ester of aN-α-amino protected amino acid and an acid under esterifying conditions,wherein the acid is carbonic acid, lower alkyl mono ester of carbonicacid, carbonic acid monoamide, N-lower alkyl carbonic acid amide, N,N-diloweralkyl carbonic acid monoamide or trimethylacetic acid.
 91. Theprocess according to claim 89 wherein the anion of the ionic fluoridesalt is (HF)_(z)F^(⊖), wherein z is 1-10.
 92. The process according toclaim 89 wherein the anion of the ionic fluoride salt is HF₂ ⁻, H₂F₃ ⁻,H₃F₄ ⁻, or Snφ₃F₂ ⁻, wherein φ is phenyl.
 93. A process for forming anamino acid fluoride in situ comprising reacting under fluorinationconditions a fluorinating effective amount of an ionic fluoride saltwith an active ester of an N-∝-amino protected amino acid, underconditions effective to form said amino acid fluoride, wherein the anionof the ionic fluoride salt has the formula: Ly(HF)_(z)F⁻, TG₁G₂G₃F₂ ⁻ orLF^(⊖); wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; y is 0 or 1, L isTG₁G₂G₃G₄; T is a Group IV element selected from the group consisting ofSi, Ge, Sn and Pb; and G₁, G₂, G₃ and G₄ are independently halogen,hydrogen, alkyl, aryl, arylalkyl, cycloalkyl or cycloalkylalkyl.
 94. Theprocess according to claim 93 wherein the active ester is an N-α-hydroxysuccinimide of N-α-amino protected amino acid, N-hydroxy piperidineester of N-α-amino protected amino acid or N-α-hydroxy-phthalimide esterof N-α-amino protected amino acid.
 95. The process according to claim 93wherein the active ester has the formula: (W)m₁Ar O—AA₁—BLK₁

wherein R₁ and R₂ taken together with the carbon atoms to which they areattached form a heteroaryl ring or aryl ring wherein said heteroarylring is an oxygen, sulfur or nitrogen containing heteroaromatic havingfrom 3 and up to a total of 13 ring carbon atoms, said heteroaryl oraryl may be unsubstituted or substituted with lower alkyl or an electrondonating group; Y is O, NR₄, CR₄R₅; R₄ and R₅ are independently hydrogenor lower alkyl; X is CR₆R₇ or NR₆; R₆ and R₇ are independently hydrogenor lower alkyl or R₆ and R₇ taken together form an oxo group or when Qis not present R₄ and R₆ taken together may form a bond between thenitrogen or carbon atom of Y and the nitrogen or carbon atom of X; Q isCR₈R₉ or NR₈; R₈ and R₉ are independently hydrogen or lower alkyl or R₄and R₈ taken together may form a bond between the ring carbon ornitrogen atom of Q and the ring carbon or nitrogen atom of R₈; and Ar isaryl, R₃ is BLK₁—AA₁, BLK₁ is an N-α-amino protecting group, AA₁ is anamino acid residue, W is an electron withdrawing group; and m₁ is aninteger from 1-5.
 96. The process according to claim 95 wherein aryl isphenyl.
 97. The process according to claim 93 wherein the anion of theionic fluoride salt is (HF)_(z)F^(⊖), wherein z is 1-10.
 98. The processaccording to claim 93 wherein the anion of the ionic fluoride salt isHF₂ ^(⊕), H₂F₃ ⁻, H₃F₄ ⁻ or Snφ₃F₂ ⁻, wherein φ is phenyl.